(1) Field of the Invention
The invention relates to a process for the reduction of free fatty acids (FFA) and preferably, cholesterol and melting point in animal fats, particularly anhydrous milkfat. The process uses .beta.-cyclodextrin and an aqueous liquid formula consisting of alkali metal hydroxide (Na or K) as a neutralizing agent, alkaline earth metal (Ca or Mg salts) as fatty acid acceptors, and preferably low melting point vegetable oils. In particular, the present invention relates to a process wherein a mixture of animal fats with the liquid formula with mild heating is used to precipitate the FFA, to decrease the melting point, and to clathrate the cholesterol with the .beta.-cyclodextrin and then the mixture is centrifuged to remove the insoluble salts of fatty acids and clathrate. The invention particularly addresses the problem of selectively removing the FFA from anhydrous milkfat without precipitating the lipid materials (the anhydrous milkfat) or damaging the fine volatile flavor components of anhydrous milkfat, which are the lactones.
(2) Description of the Prior Art
It has long been known, that very high serum cholesterol levels, high blood pressure, and an abnormal electrocardiogram (EKG) are important contributing factors to heart attacks. It is important to note that these factors become apparent long before the effects of smoking, obesity, lack of exercise are observed. The importance of serum cholesterol levels has been strengthened over the years, and one of the most consistent findings in cardiovascular studies is that high levels of plasma cholesterol are associated with atherosclerosis and enhanced risk of coronary heart disease (CHD). This effect usually is mediated through the plasma low density lipoproteins (LDL), which are the most atherogenic lipoproteins (Grundy, S. M., Am. J. Clin. Nutr. 45:1168 (1987)).
The major causes of high serum cholesterol levels are genetic disorders, heterozygous familial hypercholesterolemia (FH), and the habitual diet high in saturated fat-calories-cholesterol.
Health experts and physicians generally agree that the dietary management is the initial step in the treatment of hypercholesterolemia and hyperlipidemia. This applies even when later drug therapy is required. Changes in diet, serum cholesterol and CHD in immigrant populations have provided convincing evidence that diet plays a major role (Dyerberg, J., Nutrition Review 44(4):125 (1986)).
Although the consumption of cholesterol does not seem to be a major factor in CHD, Khosla, P., and K. C. Hayes, Biochim Biophys Acta 1210:13 (1993) reported that excessive intake of dietary cholesterol exerts a synergistic effect on the metabolism of C.sub.16:0 -rich fats, causing them to be hypercholesterolemic. In the absence of dietary cholesterol and in individuals with normal lipoprotein profiles, C.sub.16:0 does not ordinarily raise total plasma cholesterol concentration or LDL (Hayes, K. C., Food Technology Journal 50(4):92-97 (1996). Further, it has been reported that oxides of cholesterol are toxic and cause degeneration of aortic smooth muscle cells in tissue culture and may lead to the development of atherosclerosis.
Milkfat is stable to oxidation and possesses a uniquely pleasing flavor not found in other fats. Milkfat has received most attention because of its commercial importance. It confers distinctive properties on dairy products that affect processing. Milkfat is a good source of essential fatty acids and it contains a high proportion of short chain fatty acids which contributes to its ease of digestibility (Kennedy, J., Food Technol. 11:76 (1991)). Moreover, milkfat contains conjugated linoleic acids (CLA) recognized for their potential ability to inhibit cancer (Yeong et al., J. Agric. Food Chem, 37:75-81 (1989)). CLA are unusual because they are abundant in products from ruminant animals. They are formed during the process of biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen of cows and subsequently find their way into milk (Gurr, M. I., Advanced Dairy Chemistry, Lipids, P. F. Fox (ed.), p. 349. Chapman & Hall, London (1994)). One epidemiological study compared dietary habits in rural Finland which has one-quarter the incidence of colon cancer compared with urban Copenhagen, Denmark (MacLennan, R., et al., Am. J. Clin. Nutr. 31:S239 (1978). The community with a low incidence of colon cancer consumed more potatoes and whole milk than the high incidence group and ate less white bread and meat. Milkfat has a high proportion of saturated fatty acids, mainly C.sub.16:0 (26.3%), and cholesterol (0.2-0.4%) which has resulted in its decreasing consumption. This is because of the perception that the milkfat is bad for the diet.
Among most of the natural fats, milkfat is the most varied in its chemical characteristics and functional property. The melting point of milkfat increases with increasing saturation and chain length of its fatty acids components (Walstra, P., et al., Advanced Dairy Chemistry, Lipids (P. F. Fox (ed) Chapter 5, pp. 179-212 Chapman & Hall, London (1994)). The melting point of milkfat is also affected by the positioning of the fatty acid residues over the glycerol molecule (Walstra et al., IBID). In its native form, milkfat does not always suit various food formulations. For example, the wide melting range of milkfat, -40 to 40.degree. C. (Walstra et al., IBID), makes it difficult to produce spreadable butter at refrigeration temperature which is considered by many modern consumers to be an undesirable attribute. Therefore, new fields of use of milkfat are constrained due to its limited functionality (pourability and spreadability).
An optimum fat cannot always be obtained from nature. Animal fats, when viewed in their native state, have limited use. But they can become an economic asset when viewed as a raw material to produce fats with desirable health characteristics.
The fats and oils industry is looking at new techniques to alter the fat molecules. The biggest challenge is to reconcile the functional needs with the nutritional concerns. In terms of physical and nutritional performance, interesterificaton of milkfat or milkfat/vegetable oils is a useful technique to achieve a desired softening point. Interesterification of milkfat alters the distribution of fatty acids in the triacylglycerol and thus, changes its physical properties such as melting behavior, crystallization, and plasticity. Christophe, A. M., et al., Arch. Int. Physiol. Biochem 86:413 (1978) have shown that interesterification of milkfat with chemical catalysts reduces its potency to raise the blood serum cholesterol in human. Interesterified milkfat appears to be more rapidly hydrolyzed by pancreatic lipases in vitro than native milkfat (Christophe, A. M., et al., Arch Int. Physiol. Biochim. 89:B156 (1981)).
Interesterification can be accomplished by heating the fat or a blend of fat and oil in the presence of a chemical catalyst at relatively low temperature (50.degree. C.) for 30 minutes (Eckey,, E. W. Ind. and Eng. Chem. 40:1183 (1948)). Catalysts are commonly used to allow the reaction to be completed in a short time at lower temperatures. Alkali metals and alkali metal alkylates are effective low-temperature catalysts, with sodium methoxide being the most used. Directed interesterification, where the fat is heated just below its melting point, is a useful technique to remove the saturated fatty acids from the fat as crystallized trisaturated glycerides precipitates (Eckey, E. W., Ind. and Eng. Chem. 40:1183 (1948)) and therefore, improving its nutritional properties (saturated:unsaturated fatty acids ratio). Eckey was able to remove 19% trisaturated glycerides from cottonseed oil, which contains 25% saturated fatty acids. Directed interesterification had been commonly used in the industry to improve the quality of lard (Hawley, H. K., and G. W. Holman, J. Am. Oil Chem. Soc. 33:29 (1956)).
Nevertheless, interesterification has not yet been applied to the milkfat industry, since its feasibility is restricted by the fact that it is often deleterious to milkfat flavor, refining and deodorization to remove milkfat FFA (Frede, 1991; Bulletin of the International Dairy Federation No 260/1991). FFAs consume the catalyst or inactivate the active catalyst once it is formed. Sreenivasan, B., J. Am. Oil Soc. 55:796 (1978) reported that an acid value (A.V.) of 0.1 is able to poison 0.1 lb of sodium methoxide per 1000 lbs of oil. Thus, removing the FFA from anhydrous milkfat is an important step, before interesterification. Moreover, FFAs are more prone to oxidation than esterified fatty acids and hence can predispose milkfat to oxidative rancidity characterized by off-flavor described as "bitter".
U.S. Pat. No. 5,382,442 to Perlman et al. (1995) describes a blending process to increase the oxidative stability of vegetable or fish oils and animal fats. The fat blends consist of vegetable or fish oil and cholesterol reduced animal fats which comprises about 2 parts and about 9 parts linoleic acid per 1 part myristic acid.
Refining and deodorization of fats and oils are very commonly used techniques in the fat and oil industry to remove FFA. Alkali refining, used by the vast majority of European and American refiners (Braae, B., J. Am. Oil Chem. Soc 53:353 (1976); Carr, R. A., J. Am. Oil Chem. Soc. 53:347 (1976)), consists of heating the fat or oil to 75-90.degree. C. then treating it with a concentrated caustic solution of sodium hydroxides, 12 to 18.degree. Be', depending on the type of oil (cotton, soybean, corn, palm, safflower, peanut) for 30 seconds (Short-Mix process) or 0.2 seconds with 28.degree. Be' sodium hydroxide (Ultra-Short-Mix process). Using these processes for milkfat is very detrimental to the lactones, the major milkfat flavor components. Lactones (.gamma. or .delta.) are cyclic esters of .gamma. or .delta.--hydroxy acid which in the presence of concentrated caustic solution are rapidly hydrolyzed to give the open chain salt of hydroxy acids. Consequently, the prior-art processes, short reaction time, high concentration caustic solution, and high temperature cannot be applied to milkfat.
Deodorization, very commonly used in the fats and oils industry, consists of blowing steam through hot oil at 200.degree. to 275.degree. C. under a high vacuum (3-10 torr). The deodorization process removes simultaneously the FFAs, fat soluble vitamins (A, E, D, K), monoglycerides, sterols, and some pigments such as caratenoids. As the term implies, deodorization strips off the aroma and flavors of fats and oils resulting in a bland finished product which is viewed as extremely undesirable for milkfat. Therefore, refining by using concentrated alkali metal hydroxide and deodorization of milkfat reduces the FFA losing the volatile fine milkfat flavor, the aroma, and the vitamins content. This puts milkfat in the same class as other cheap raw materials.
U.S. Pat. No. 3,560,219 to Attebery describes the use of metal salts under alkaline conditions to precipitate dissolved lipids in an aqueous food product such as cheese whey. This process however, is not selective for free fatty acids since all the lipid materials in the food products were precipitated. Therefore, this process cannot be used for food products consisting of pure lipid such as anhydrous milkfat.
Thus, the prior art has recognized the need to remove the FFA from milkfat without precipitating the milkfat per se or damaging the fine volatile milkfat flavor components. The growing concern regarding the cholesterol content of the human diet has led the food processors to develop several techniques to reduce cholesterol from milkfat.
Reduction of Milkfat Cholesterol by Enzymes
Cholesterol reductase catalyzes the conversion of cholesterol in the presence of NADPH to coprostanol which passes through the body without being absorbed (MacDonald, I. A., et al., J. Lipid Res. 24:675 (1983); the success-to-date is limited. In milk treated with cholesterol oxidase, the cholesterol concentration was reduced by 78% within 3 hours at 37.degree. C. (Smith, M., et al., Journal of Agricultural and Food Chemistry, 39:2158 (1991)). It has been reported that oxides of cholesterol are themselves toxic (Peng, S.-K, and R. J. Morin, Biological Effects of Cholesterol Oxides. CRC Press, Boca Raton, Ann Arbor, London 1991). These processes are not practical for anhydrous milkfat.
Short-Path Distillation (SPD)
SPD consists of evaporation of molecules into substantially gas-free space, e.g., vacuum. The control factor is the rate at which the molecules escape from the heated surface of the distilling liquid and are received by the cooled condenser surface. Stripping vitamins A and E, sterols and volatiles from natural oils and separation of mono- and diglycerides and fatty acids are uses related to food besides chemical and pharmaceutical areas. Arul et al., J. Am. Oil Chem Soc. 65:1642 (1988) fractionated milkfat by SPD into four fractions at temperatures of 245 and 265.degree. C. and pressures of 220 and 100 mm Hg. Two fractions were liquid, one fraction was semi-solid, and one fraction was solid at room temperature. The solid fraction contains cholesterol at a concentration of 0.2 mg/g fat compared with 2.6 mg/g fat in native milkfat or to 16.6 mg/g fat in the liquid fraction. SPD of milkfat offers an opportunity to obtain fractions from milkfat with different chemical and physical properties. However, there are some major drawbacks of this technique: (1) the low yield fraction where the cholesterol reduction was observed is solid and therefore, has a limited functionality; (2) the use of high temperature can decompose or polymerize the triacylglycerol, particularly those with high unsaturation even when distilled under vacuum; and (3) high capital investment.
Supercritical Fluid Extraction (SFE)
In this process, a product is treated with gas (usually carbon dioxide) of high density, low viscosity, and reduced surface tension under high pressure and temperature. The technique has been applied for delipidation of protein and reduction of cholesterol from different foods, decaffeination of coffee and tea, and also removal of bitter aroma compounds from hops. The procedure has the advantage of the absence of potentially toxic solvents and no toxic by-products are formed (Friedrich, J. P. and E. H. Pryde, J. Am. Oil Chem. Soc. 61:223 (1984)). Arul et al., J. Food Sci. 52:1231-1236 (1987) fractionated milkfat into 8 different fractions at 10-35 MPa and 50 to 70.degree. C. They found that the cholesterol tended to concentrate in the low and intermediate melting fractions. Lim, S. and Rizvi, S. S. H., J. Food Science 61(4):817-821 (1996) achieved an overall cholesterol reduction of 92.6% with a process yield of 88.5%. The extraction was done at 40.degree. C. and 24.1-27.5 MPa. The indiscriminate solvency of supercritical carbon dioxide is a major drawback in cholesterol reduction, since some triacylglycerols were extracted along with cholesterol which can disrupt the normal aromatic balance of milkfat.
Vacuum Steam Distillation
Vacuum steam distillation for deacidification and deodorization of oils has been practiced in Europe for many years. The technique consists of blowing superheated steam through hot oil at 200.degree.-275.degree. C. under high vacuum. General Mills, Inc. (Minneapolis, U.S.A.) has disclosed a vacuum steam distillation process for simultaneous cholesterol reduction and deacidification of butter oil (Marschner and Fine, U.S. Pat. No. 4,804,555 1989). The cholesterol removal achieved by this technique was 90%, with a 95% yield. The major drawback of this technique is that the heated steam strips off the volatile flavor components of milkfat along with cholesterol and FFA. The loss of the fine butter flavor puts milkfat in the same class as other cheap fats.
Complex Formation
This technique is used to reduce the cholesterol in milk and dairy products by complexing the cholesterol and its esters with a complexing agent such as .beta.-cyclodextrin (.beta.-CD).
Cyclodextrins are cyclic oligosaccharides obtained by enzymatic degradation of starch. They consist of six, seven, or eight glucose monomers arranged in a donut shaped ring, which are denoted alpha, beta or gamma cyclodextrin, respectively. .beta.-CD are not hygroscopic and contain 13.6% moisture at 30.degree. C. and 86% relative humidity (RH) (Szejtli, J., et al, Inclusion Compound 3:331 (1984)). Cyclodextrins are water soluble due to the location of free hydroxyl groups on the external rim of the molecule (Szejtli, J., et al, Inclusion Compound 3:331 (1984)). Solubility is a function of temperature. The higher the temperature the higher the solubility. The solubility of .beta.-CD increases from 0.8% at 0.5.degree. C. to 39.7% at 90.degree. C. The internal cavity which is hydrophobic allows the cyclodextrins to complex molecules such as aromatic alcohols, fatty acids and their esters and cholesterol. .beta.-CD has been used to reduce cholesterol in milkfat for several reasons:
1--The relative size and geometry of the .beta.-CD internal cavity allowed good complexing with free and esterified cholesterol; PA1 2--The realization of industrial scale production of .beta.-CD; PA1 3--The intensive research on toxicity of .beta.-CD during the past decade, has assured its safety as a food ingredient.
Currently, cyclodextrins are used: (1) to control volatility of agricultural compounds which control pathogens, insects, and weeds; (2) in pharmaceutical products (drugs, vitamins), fragrance, and skin care lotions to improve stability by means of encapsulation; and (3) to enhance color, odor, and flavor stability in beverages and processed foods (Szejtli, J., Inclusion Compound 3:311 (1984)).
The most important parameters that determine whether a given molecule can form complexes are its hydrophobicity, relative size and geometry in relation to the cyclodextrin cavity (Szejtli, J., Inclusion Compound 3:331 (1984)). When dissolved in water, the cyclodextrin molecules are able to accommodate smaller guest molecules, or functional groups of molecules less hydrophilic than water in their internal cavities (Szejtli, J., Inclusion Compound 3:331 (1984)). In aqueous solution, the slightly apolar cyclodextrin cavity is occupied by water molecules, an energetically unfavored process (polar-apolar interaction). These water molecules are therefore readily substituted by appropriate guest molecules "such as cholesterol or FFA and their esters" which are less polar than water (Szejtli, J., Inclusion Compound 3:331 (1984)).
Six-month oral chronic toxicity of .beta.-CD (Szejtli, J., Inclusion Compound 3:331 (1984)) was studied in rats by feeding up to 1.6 g/body weight kg/day and up to 0.6 g/body weight kg/day in dogs. Weight gain, food consumption, and clinico-biochemical values were not affected. ".beta.-CD showed no embryo-toxic effect. Orally administered .beta.-CD can thus be considered a non-toxic substance (Szejtli, J., Inclusion Compound 3:331 (1984)).
U.S. Pat. No. 4,880,573 (1989) to Courregelongue et al. describes the use 10% .beta.-CD to remove 41% of the sterols from anhydrous milkfat. EP-A1-0 326 469 (1989) (European Patent) to Bayol et al. indicated the removal of 80% .DELTA..sup.4 -cholesten-3-one from anhydrous milkfat. U.S. Pat. No. 5,232,725 to Roderbourg et al.(1993) describes a process for the removal of 37% or more of the cholesterol content of animal fat along with free fatty acids in one single operation using .beta.-CD as a complexing agent. U.S. Pat. No. 5,264,241 to Graille et al.(1993), using .beta.-CD, reported the simultaneous removal of 50% cholesterol and 52% FFA from cream. U.S. Pat. No. 5,223,295 to Maffrand et al. describes a process for removing cholesterol from fats of animal origin by complexing the steroidal compounds by means of a cyclodextrin, in an aqueous medium, under agitation for a period of 5 hours. This process is not only relatively long but it permits a limited reduction of the cholesterol content in a single operation. U.S. Pat. No. 4,980,180 (1990) to Cully et al. describes a method for removal of .beta.-CD from egg materials using .beta.-amylases. The patent recognizes the problem of incomplete removal of the cyclodextrins. In Australia, Okenfull et al., (1991)(PCT W091/11114) invented a process called SIDOAK to reduce cholesterol in dairy products. The process consists of adding .beta.-CD to milk and mixing below 10.degree. C. The insoluble complexes of cholesterol and .beta.-CD were removed by centrifugation. The maximum cholesterol reduction obtained was 80-90%. Yen, C. G., and L. J. Tsai, J. Food Sci. 60:561 (1995), using 10% .beta.-CD, indicated the removal of 95% cholesterol simultaneously with 50% FFA from lard. U.S. Pat. No. 5,484,624 to Awad et al. (1996) used .beta.-CD to reduce the cholesterol in egg yolk by 96%. The process consists of mixing a diluted egg yolk (9&lt;pH&lt;10.5) with .beta.-CD at 50.degree. C. for 10 minutes. The complexes .beta.-CD-cholesterol were removed from the medium by centrifugation.
The overall conclusion from all the .beta.-CD processes developed to reduce cholesterol and FFA in animal fat is that there is a need for improvement. The modification of anhydrous milkfat by interesterification to improve its nutritional and functional properties remains constrained due to the high FFA content which poisons the catalysts.
Thus, the prior art has recognized that modifying milkfat by interesterification or blending and reducing its cholesterol content will not be competitive in the manufacture of fat products, at least as long as no process has been developed to simultaneously reduce cholesterol and FFA without damaging the volatile fine flavor components of milkfat, particularly the lactones. The wide melting range of milkfat, -40 to 40.degree. C. (Walstra et al., IBID), makes it difficult to produce spreadable butter at refrigeration temperature which is sought by many modern consumers as an undesirable attribute. Therefore, new fields of applications of milkfat remain constrained due to its limited functionality (pourability and spreadability). The fractionation of milkfat by physical processes, into different fractions, has been effective only to a modest extent in improving its melting property. This known process, however, is tedious, time consuming and expensive. Another disadvantage associated with fractionation is the disruption of the normal aromatic balance of the milkfat. Therefore, the prior art has recognized the need for a process which produces a soft milkfat with a low melting point so that it is spreadable at refrigeration temperatures.